CAJ | 학술논문

应用基于RNA干扰（ RNA interference，RNAi）原理的大豆转基因技术，能够在转录水平沉默同源靶基因，这为转基因抗病毒作物的培育提供了新的策略。通过比对已报道的能与病毒VPg发生互作的10种植物的eIF4E氨基酸序列，确定出大豆eIF4E的保守区间为62～237位氨基酸序列，克隆出406 bp的干扰片段eIF4Ei（含58 bp特异性重组序列attB），进而采用GATEWAY技术构建了干扰表达载体pB7GWIWG2（Ⅱ）-eIF4Ei。之后通过测序证实干扰片段eIF4Ei在载体构建过程中没有发生变异；酶切试验证实终端质粒的2个ccdB位点均被eIF4Ei置换；用启动子和终止子设计的引物对含有重组质粒的菌液进行PCR扩增，证实2个干扰片段以相反的方向插入到终端质粒中。从而证明反向重复的干扰表达载体构建成功，为应用基于RNAi原理的大豆转基因技术培育对大豆花叶病毒具有抗性的大豆新种质提供了基础材料。
응용기우RNA간우（ RNA interference，RNAi）원리적대두전기인기술，능구재전록수평침묵동원파기인，저위전기인항병독작물적배육제공료신적책략。통과비대이보도적능여병독VPg발생호작적10충식물적eIF4E안기산서렬，학정출대두eIF4E적보수구간위62～237위안기산서렬，극륭출406 bp적간우편단eIF4Ei（함58 bp특이성중조서렬attB），진이채용GATEWAY기술구건료간우표체재체pB7GWIWG2（Ⅱ）-eIF4Ei。지후통과측서증실간우편단eIF4Ei재재체구건과정중몰유발생변이；매절시험증실종단질립적2개ccdB위점균피eIF4Ei치환；용계동자화종지자설계적인물대함유중조질립적균액진행PCR확증，증실2개간우편단이상반적방향삽입도종단질립중。종이증명반향중복적간우표체재체구건성공，위응용기우RNAi원리적대두전기인기술배육대대두화협병독구유항성적대두신충질제공료기출재료。
Soybean transformation based on RNA interference ( RNAi) can specifically silence the homologous target genes in mRNA level ,providing a new strategy for disease-resistant breeding .In this study ,we made the ami-no acid sequence alignment of eIF4E from ten different kinds of plants which have been identified to interact with the virus VPg ,then we determined the conserved interval of soybean eIF 4 E was 62-237 amino acid sequence and cloned the 406 bp interference fragment named eIF4Ei (including 58 bp specific recombination sequence attB ).U-sing the GATEWAY technology ,the RNAi vector pB7GWIWG2(Ⅱ)-eIF4Ei was constructed,which was identified by PCR amplification ,sequencing and restriction digestion .The identification of the recombinant expression vector ensured the eIF4 Ei interference fragment no mutation in the process of vector construction and two ccdB sites were all replaced by eIF4Ei,in addition,the insert direction of the two interference fragments in the terminal plasmid was conversed.Overall,the RNAi vector was successfully constructed to be an inverted repeat structure ,providing the basic materials for cultivating new germplasm of soybeans resistant to SMV using the soybean transformation technol -ogy based on RNAi .